Peptide gels formed of self-assembling peptide derivatives are known to be useful in the fields of regenerative medicine and surgery (Patent Literature 1). Peptide derivatives represented by the structural formulae: Ac-Arg-Leu-Asp-Leu-Arg-Leu-Ala-Leu-Arg-Leu-Asp-Leu-Arg-NH2 (SEQ ID NO: 1) and Ac-Arg-Leu-Asp-Leu-Arg-Leu-Leu-Leu-Arg-Leu-Asp-Leu-Arg-NH2 (SEQ ID NO:2) (wherein Ac represents an acetyl group; the same applies hereinafter) are disclosed therein as peptides having the most preferred amino acid sequences with terminal modifications.
The applications of the self-assembling peptide derivatives and gels thereof include, for example, substrates for cell culture; cosmetics such as skin care products and hair care products; medical products such as decubitus preparations, bone fillers, injectable agents for aesthetic, adjuvant to ophthalmic operation, artificial vitreous bodies, artificial lenses, joint lubricants, ophthalmic solutions, DDS substrates, and hemostats; water retention materials for moistening; desiccants; and coating agents for medical devices such as contact lenses (Patent Literature 1).
Synthetic processes for peptides are generally known to include chemical synthesis, enzymatic synthesis and recombinant synthesis.
Enzymatic synthesis has the advantages of being performed under mild reaction conditions and forming few by-products, and other advantages. Known methods of enzymatic synthesis utilize, for example, metallopeptidases (such as thermolysin), aminoacyl-tRNA synthetases, alanine ligases, nonribosomal peptide synthetases, or the like. However, enzymatic synthesis has some problems. For example, some of the enzymes used in conventional enzymatic synthesis are at high cost. In addition, the desired peptide design is not always achieved due to preferential substrate specificity of enzymes.
Recombinant synthesis is a process in which DNA containing a nucleotide sequence encoding a peptide of interest is prepared and then expressed in host cells or the like. The vectors used for gene expression and peptide synthesis are, for example, plasmids, baculovirus, or the like, which are selected depending on the type of cells to be used. However, recombinant synthesis also has some problems. For example, the vectors into which DNA has been introduced sometimes do not produce the exact peptide of interest.
Chemical synthesis is a process in which amino acids are chemically coupled stepwise one by one. Chemical synthesis is generally known to include liquid-phase synthesis and solid-phase synthesis. Solid-phase peptide synthesis is a process using a solid phase, for example, polystyrene polymer gel beads about 0.1 mm in diameter having an amino group attached to the surface, and from the attached amino group, an amino acid chain is elongated by one amino acid each time through dehydration reaction. At the end of the synthesis of the desired peptide sequence, the peptide is cut off from the solid-phase surface to give a substance of interest. Solid-phase peptide synthesis allows the synthesis of ribosomal peptides, which are difficult to synthesize in bacteria, the incorporation of non-natural amino acids such as D-amino acids and heavy atom-substituted amino acids, the modification of peptide/protein backbone, and the like.
Solid-phase peptide synthesis is, however, inefficient because amino acids are coupled one by one at each synthesis step, and is also not economical because carriers for solid-phase synthesis are expensive and the amounts of reagents used are large. Further, scale-up of facilities is also difficult.
The peptide derivatives represented by the structural formulae: Ac-Arg-Leu-Asp-Leu-Arg-Leu-Ala-Leu-Arg-Leu-Asp-Leu-Arg-NH2 (SEQ ID NO: 1) and Ac-Arg-Leu-Asp-Leu-Arg-Leu-Leu-Leu-Arg-Leu-Asp-Leu-Arg-NH2 (SEQ ID NO:2), as disclosed in Patent Literature 1, are produced by conventional solid-phase synthesis. Hence there is a demand for the development of an efficient production process that allows reduction in the production cost of the peptide derivatives and scale-up of the production.
Each of the peptide derivatives, Ac-Arg-Leu-Asp-Leu-Arg-Leu-Ala-Leu-Arg-Leu-Asp-Leu-Arg-NH2 (SEQ ID NO: 1) and Ac-Arg-Leu-Asp-Leu-Arg-Leu-Leu-Leu-Arg-Leu-Asp-Leu-Arg-NH2 (SEQ ID NO:2), disclosed in Patent Literature 1 has four Arg residues (basic amino acid residues) and two Asp residues (acidic amino acid residues) within the sequence, and accordingly the peptide derivatives can be made into various salt forms. It is generally known that depending on the salt form of a peptide derivative, the functions and properties of the peptide derivative will change. Based on this, there is also a need for searching an appropriate salt form of the above peptide derivatives in order to satisfy the existing demands that the peptide derivatives should have, in addition to the function of dissolving in water to form a high strength peptide gel, certain properties such as easy handling and storage stability that are suitable for commercial production.